Eriocheiris Infection Research Articles

Comprehensive analysis of the mRNA and miRNA transcriptome implicated in the immune response of Procambarus clarkii to Spiroplasma eriocheiris

In recent years, the red swamp crayfish (Procambarus clarkii, P. clarkii) farming industry has suffered huge economic losses due to the pathogenic bacterium Spiroplasma eriocheiris (S. eriocheiris). To elucidate the immune response mechanism and identify hub immune genes as well as their associated microRNAs that regulate the host response of P. clarkii against S. eriocheiris infection, we conducted a comprehensive analysis on P. clarkii hemocyte mRNA and microRNA (miRNA) transcriptomes at different infection stages using third- and second-generation sequencing technologies. In full-length transcriptome functional annotation, 8155 unigenes were annotated, and 1168 potential new transcripts were predicted. In the mRNA transcriptome, a total of 3168 differentially expressed genes were identified at different infection stages, including 1492 upregulated and 1676 downregulated genes (duplicate genes excluded). Transcriptome analysis revealed 880 differentially expressed genes involved in multiple pathways and processes such as endocytosis, autophagy, lysosome, mTOR signaling, phagosome, and the Fanconi anemia pathway. Mfuzz analysis was employed to integrate and cluster the differential expression trends of genes across the three infection stages. In the miRNA transcriptome, 234 miRNAs and 966 predicted target genes were identified, with 86 differentially expressed miRNAs identified across the three time periods. A significant difference (P < 0.05) was observed for miRNAs including pcl-miR-146-3p, pcl-miR-74-3p, pcl-miR-225-5p, and pcl-miR-68-5p. These miRNAs are involved in multiple immune and autophagy-related pathways and have regulatory effects on immune genes including Vps26, lqf, and ERK-A. Based on the differentially expressed immune-related genes, we constructed a protein-protein interaction (PPI) network, which revealed the interactions among hub genes including Rac1, Akt1, Rho1, and Egfr. We also constructed a miRNA-gene interaction network in immune and autophagy-related processes, highlighting the potential regulatory effects of miRNAs including pcl-miR-183-5p, pcl-miR-146-3p, pcl-miR-176-5p, and pcl-miR-225-5p on proteins including LST8, SNAP29, Rab-7A, and ERK-A. To conclude, this study has identified hub immune genes and corresponding regulatory miRNAs in P. clarkii hemocytes in response to S. eriocheiris infection and explored the roles of these genes in selected pathways and processes. These findings are expected to provide further insights into the molecular mechanisms that confer resistance to S. eriocheiris infection in P. clarkii.

Single-cell RNA-sequencing reveals Eriocheir sinensis hemocyte subpopulations and their molecular responses to Spiroplasma eriocheiris infection

The hemocytes is the central part of the Eriocheir sinensis innate immune system, however, their subclasses have not been completely defined and the single-cell gene transcriptional profiles and functions are still unknown. In the present study, the single-cell mRNA sequencing (scRNA-seq) was carried out to build a single-cell gene expression map of E. sinensis hemocytes and further analysis of the interaction molecular mechanism between the crab and novel aquatic pathogens Spiroplasma eriocheiris. By scRNA-seq, we successfully identified nine cell clusters (Hem1–9) and predicted their functions according to the specific marker genes. Pseudotime inference analysis revealed that all the identified cells undergo three stages, state 1 (the initial point), state 2 and state 3 (the other two branches). After being infected by S. eriocheiris, lots of different expression genes (DEGs) were identified in each cell cluster compared with the control group (with fold change ≥2 and p-value <0.05). The bioinformatics analysis found that the Hem1 and Hem5 were the main cell clusters synthesis and release of antimicrobial peptides (AMPs) for help the host against the pathogen infection. The Hem5 also could activate the proPO system to participate in the process of host eliminate the invading S. eriocheiris. Hem7, Hem8, and Hem9 were mainly responsible for killing the invading pathogen through phagocytosis. In particular, there were also many immune-related genes identified, including β-glucan binding protein (BGBP), Clip-domain serine proteases (CLIPs), phenoloxidase 1 (PO1), anti-lipopolysaccharide factors (ALFs), syndecan, and lysozyme C, which not only played critical roles in the crab immune system but also could serve as the marker genes for identifying cell subgroup. In conclusion, the results obtained in the present study could provide pivotal insights into the crab hemocytes cell-type-specific molecular mechanisms response S. eriocheiris infection at a single-cell level.

Multi-omics characterization and identification of small non-coding RNAs in Spiroplasma eriocheiris

Spiroplasma eriocheiris is the major pathogen in the aquaculture shrimp and crab tremor disease; its infection causes heavy losses in aquaculture. Previous investigations concentrated on how the host responds to S. eriocheiris infection, with little known on the regulation of virulence and pathogenesis by the bacteria themselves. A body of increasing evidence shows that bacterial small noncoding RNAs (sRNAs) play a crucial part in regulating bacterial virulence and pathogenesis; however, whether there are some sRNAs in S. eriocheiris and how those sRNAs regulate virulence and pathogenesis are still unclear. This study analyzed multi-omics data integration to identify the potential sRNAs and their mediated regulatory network in S. eriocheiris. First, through an integration analysis of the public genomic and transcriptomic data, 54 potential sRNAs were identified in S. eriocheiris with a 50–250 nt length distribution and the base composition mainly AT. Next, sRNA profiling of the in vitro logarithmic and decline phase of S. eriocheiris, as well as the early and onset stage of infection in Eriocheir sinensis, was performed, showing that 4 sRNAs (SR01, SR05, SR08, and S04) were both down-regulated in the logarithmic and the onset stage of infection. Subsequently, integrated with sRNAs target gene prediction and target gene expression analysis, a regulatory network composed of four sRNAs and 96 target genes was constructed. Last, an RNA pull-down experiment was performed on the SR01, which exhibited the highest abundance and the highest number of target genes, resulting in 37 highly credible target genes with multiple genes related to bacterial virulence, such as GTPases and virulence factor coding genes. Collectively, this study identified the sRNAs in S. eriocheiris and revealed their mediated regulatory network for regulating virulence and pathogenesis, thus paving a new avenue for disease prevention and control in aquaculture.

Hemolymph exosomes inhibit Spiroplasma eriocheiris infection by promoting Tetraspanin-mediated hemocyte phagocytosis in crab.

Exosomes released from infected cells are thought to play an important role in the dissemination of pathogens, as well as in host-derived immune molecules during infection. As an intracellular pathogen, Spiroplasma eriocheiris is harmful to multiple crustaceans. However, the immune mechanism of exosomes during Spiroplasma infection has not been investigated. Here, we found exosomes derived from S. eriocheiris-infected crabs could facilitate phagocytosis and apoptosis of hemocytes, resulting in increased crab survival and suppression of Spiroplasma intracellular replication. Proteomic analysis revealed the altered abundance of EsTetraspanin may confer resistance to S. eriocheiris, possibly by mediating hemocyte phagocytosis in Eriocheir sinensis. Specifically, knockdown of EsTetraspanin in E. sinensis increased susceptibility to S. eriocheiris infection and displayed compromised phagocytic ability, whereas overexpression of EsTetraspanin in Drosophila S2 cells inhibited S. eriocheiris infection. Further, it was confirmed that intramuscular injection of recombinant LEL domain of EsTetraspanin reduced the mortality of S. eriocheiris-infected crabs. Blockade with anti-EsTetraspanin serum could exacerbate S. eriocheiris invasion of hemocytes and impair hemocyte phagocytic activity. Taken together, our findings prove for the first time that exosomes modulate phagocytosis to resist pathogenic infection in invertebrates, which is proposed to be mediated by exosomal Tetraspanin, supporting the development of preventative strategies against Spiroplasma infection.

Two autophagy-related proteins, ATG5 and ATG7, from Eriocheir sinensis involved into Spiroplasma eriocheiris infection

Autophagy-related proteins (ATGs) play an important role in the immune process by regulating autophagy. Eriocheir sinensis tremor disease (TD) is the most hazardous disease caused by Spiroplasma eriocheiris. However, it is unclear whether ATGs play a role in the development of TD. In this study, the function of autophagy and ATG (ATG5 and ATG7) was investigated. Both E. sinensis ATG5 and ATG7 (EsATG5 and EsATG7) were highly transcribed in hemocytes. After S. eriocheiris infection, the transcriptions of EsATG5 and EsATG7 were significantly up-regulated in crab hemocytes. After gene silencing and S. eriocheiris infection, the mortality rate of E. sinensis and the copy number of S. eriocheiris in hemocytes were remarkably increased. These results suggested that EsATG5 and EsATG7 played an important immune role in S. eriocheiris infection. According to the result of western blot using polyclonal antibody of EsATG7, EsATG7 protein expression was increased in S. eriocheiris infection. Meanwhile, EsATG7 protein expression was considerably changed after stimulation with autophagy activator (Rapamycin, RM) and autophagy inhibitor (3-methyladenine, 3-Ma). These results shown EsATG7 was involved in autophagy. After S. eriocheiris infection, the cell activity of the RM group was significantly increased, the copy number of S. eriocheiris in RM group was decreased, while the results in the 3-Ma group were completely opposite. In conclusion, the EsATG gene through regulated E. sinensis autophagy to improve the host's immune ability to resist S. eriocheiris infection.

Screening and functional analysis of three Spiroplasma eriocheiris glycosylated protein interactions with Macrobrachium nipponense C-type lectins.

N-glycosylation, one of the main protein posttranslational modifications (PTMs), plays an important role in the pathogenic process of pathogens through binding and invasion of host cells or regulating the internal environment of host cells to benefit their survival. However, N-glycosylation has remained mostly unexplored in Spiroplasma eriocheiris, a novel type of pathogen which has serious adverse effects on aquaculture. In most cases, N-glycoproteins can be detected and analyzed by lectins dependent on sugar recognition domains. In this study, three Macrobrachium nipponense C-type lectins, namely, MnCTLDcp1, MnCTLDcp2 and MnCTLDcp3, were used to screen S. eriocheiris glycosylated proteins. First, qRT-PCR results showed that the expression levels of the three kinds of lectins were all significantly up-regulated in prawn hearts when the host was against S. eriocheiris infection. A bacterial binding assay showed that purified recombinant MnCTLDcp1, MnCTLDcp2 and MnCTLDcp3 could directly bind to S. eriocheiris in vitro. Second, three S. eriocheiris glycosylated proteins, ATP synthase subunit beta (ATP beta), molecular chaperone Dnak (Dnak) and fructose bisphosphate aldolase (FBPA), were screened and identified using the three kinds of full-length C-type lectins. Far-Western blot and coimmunoprecipitation (CO-IP) further demonstrated that there were interactions between the three lectins with ATP beta, Dnak and FBPA. Furthermore, antibody neutralization assay results showed that pretreatment of S. eriocheiris with ATP beta, Dnak and FBPA antibodies could significantly block this pathogen infection. All the above studies showed that the glycosylated protein played a vital role in the process of S. eriocheiris infection.

Protective effect of chicken egg yolk immunoglobulin (IgY) against Spiroplasma eriocheiris infection in Chinese mitten crab

The aquaculture of the Chinese mitten crab Eriocheir sinensis has become the leading and pillar of aquaculture in China. The tremor disease (TD) caused by Spiroplasma eriocheiris is the most destructive disease of E. sinensis. At present, the treatment of TD in crabs mainly depends on antibiotics. To protect the environment and the health of aquatic products, it is urgent to establish an effective and pollution-free green prevention and control technology. This study aimed to investigate the prevention and control of the TD by immunization with chicken yolk antibody (IgY). Anti-S. eriocheiris IgY was obtained by intramuscular injection of ultrasonically inactivated S. eriocheiris into chickens. After successful isolation and purification of IgY from eggs, ELISA and sensitivity tests were performed. Specific IgY was able to specifically react at a titer of 1:10,000 to antigen at a minimum concentration of 20 μg/mL and to inhibit the growth of S. eriocheiris in vitro. The affection of IgY in vivo to resist S. eriocheiris infection was demonstrated by feeding or injection of specific IgY. The copy number of S. eriocheiris in crab hemocytes with specific IgY administration was significantly less than in crabs that received normal IgY or without IgY. Meanwhile, the survival rate of E. sinensis with anti-S.eriocheiris- specific IgY using both injection and feeding methods significantly increased during S. eriocheiris infection. The electron microscope results showed that the structure of S. eriocheiris in specific IgY group was completely irregular and broken pin vivo. Finally, this study provides a reference for clarifying the potential role of specific IgY to help E. sinensis resist S. eriocheiris infection.

Eriocheir sinensis vesicle-associated membrane protein can enhance host cell phagocytosis to resist Spiroplasma eriocheiris infection

Vesicle-associated membrane protein (VAMP) belongs to the receptor protein on the membrane of the secretory transport vesicle and involves in host immune function. The intracellular pathogen Spiroplasma eriocheiris could cause Eriocheir sinensis tremor disease. In a previous study, it was found E. sinensis VAMP (EsVAMP) was differently expressed in S. eriocheiris infection by proteomics analysis. This study mainly aims at the function of EsVAMP in the process of the S. eriocheiris infection. The length of EsVAMP gene was 1681 bp, which contained a 395 bp open reading frame, 90 bp 5′-non-coding region (UTR) and 1277 bp 3′-UTR. The results of qPCR showed that EsVAMP was expressed highly in hemocytes and nerves, followed by gills, intestines and hepatopancreas, and lowly expressed in heart and muscles. EsVAMP in hemocytes was up-regulated after S. eriocheiris infection. After EsVAMP over-expression and S. eriocheiris infection, the RAW264.7 cell morphology and cell viability of the experiment group were significantly better than the control group. Meanwhile, the copy number of S. eriocheiris in the experiment group was significantly lower than that in the control group. After EsVAMP and pCMV-Cre-mCherry were ligated and transfected into RAW264.7 cells, it was found that EsVAMP and lysosome co-localized. Meanwhile, the phagocytosed inactivated S. eriocheiris number and phagocytosed efficiency in RAW264.7 cells were increased significantly. The interference experiment was carried out by synthesizing EsVAMP dsRNA to verify that the EsVAMP transcriptions were successfully suppressed. The S. eriocheiris copy number and the mortality of crab increased significantly after EsVAMP RNAi and S. eriocheiris infection. Meanwhile, the phagocytosed inactivated S. eriocheiris number and phagocytosed efficiency in hemocytes decreased significantly after EsVAMP RNAi and S. eriocheiris infection. These results showed that VAMP was involved in the cell phagocytosis to resist pathogen infection.

Oxidative stress responses of the crayfish Procambrus clarkii to Spiroplasma eriocheiris challenge

As a virulent pathogen, Spiroplasma eriocheiris influences most crustaceans, including the freshwater crayfish Procambrus clarkii. The antioxidant enzymes related to oxidative stress during the pathogenesis of S. eriocheiris in the primary target tissues of hepatopancreas and gills from P. clarkii were analyzed at various time intervals post-infection. H.E histopathology and in situ hybridization assays confirmed the S. eriocheiris infection and detected typical histological changes. Compared with the control group, catalase (CAT) activities in both tissues reduced significantly at the early time of S. eriocheiris challenge (P < 0.05), but recovered gradually at the late stage while still lower than the control group in the hepatopancreas (P < 0.05). A similar tendency was observed in the change of superoxide dismutase (SOD) activities. Acid phosphatase (ACP) and alkaline phosphatase (AKP) activities increased considerably and achieved peak values at 7 days post-infection (dpi) in the hepatopancreas and 5 dpi in the gills, but decreased significantly later till 12 dpi in both tissues (P < 0.05). Peroxidase (POD) activities and malondialdehyde (MDA) contents increased dramatically since the first day and kept a high level during the challenge course. The activities of antioxidant enzymes were generally higher in the hepatopancreas than in the gills. These results suggested that S. eriocheiris affected the antioxidative situation and caused oxidative stress and tissue damage by confusing antioxidant enzymes in P. clarkii.

Ubiquitin-modified proteome analysis of Eriocheir sinensis hemocytes during Spiroplasma eriocheiris infection

Spiroplasma eriocheiris, the pathogen of Eriocheir sinensis tremor disease (TD), has bring a huge economic loss to China aquaculture. The hemocytes of crab as the first target cells of S. eriocheiris, but the interactive relationship between the E. sinensis and this pathogen not particularly clear. The present study is the first time to analysis the role of protein ubiquitination in the process of E. sinensis hemocytes response S. eriocheiris infection. By applying label-free quantitative liquid chromatography with tandem mass spectrometry proteomics, 950 lysine ubiquitination sites and 803 ubiquitination peptides on 458 proteins were identified, of which 48 ubiquitination sites on 40 proteins were quantified as significantly changed after the S. eriocheiris infection. Bioinformatics analysis of ubiquitination different proteins suggested many biological process and pathways were participated in the interaction between S. eriocheiris and host cell, such as ubiquitin system, endocytosis, prophenoloxidase system (proPO system), cell apoptosis, glycolysis. Our study can enhance our understanding of interaction between the crab and S. eriocheiris, and also provides basis to study the role of protein ubiquitination in other crustacean innate immune system.

Leveraging lncRNA-miRNA-mRNA network to reveal anti-Spiroplasma eriocheiris infection mechanisms in Macrobrachium nipponense

The oriental river prawn Macrobrachium nipponense (M. nipponense) is a commercially important shrimp susceptible to Spiroplasma eriocheiris (S. eriocheiris) infection. Long non-coding RNAs (lncRNAs) play critical roles in a host immune defense against invading pathogens through acting as competing endogenous RNAs (ceRNAs). However, the protective roles of lncRNAs associated ceRNAs in M. nipponense against S. eriocheiris infection remain largely unexplored. To address this problem, a comprehensive lncRNA-mRNA-miRNA network in the hemocyte of M. nipponense challenged with S. eriocheiris at different time points (0, 6, 24, and 48 h) was constructed in this study. The results showed that 238 differentially expressed lncRNAs (DElncRs), 3547 DEmRNAs, and 258 DEmiRNAs were identified during the challenge process. The target genes of DEmRNAs, DElncRNAs and DEmiRNAs were significantly enriched in multiple immune-related pathways, such as Toll-like receptor, PI3K-Akt, and TNF signaling pathways. The lncRNA associated ceRNAs network was also constructed, including 12 DElncRNAs, 23 DEmiRNAs, and 25 DETmRNAs. This network revealed that DElncRNAs (transcript_18441, transcript_11191, transcript_10673, transcript_10806, and transcript_17607), DEmiRNAs (miR-2c, miR-71-3p, miR-423-5p, and miR-423a), and DETmRNAs (ALF, PCE, and PPAE) were involved in M. nipponense immune defense against S. eriocheiris challenge. Taken together, our study identified crucial lncRNA associated ceRNA networks, playing pivotal roles in the immune response of M. nipponense against S. eriocheiris infection, and thus paving a novel avenue to develop strategies for spiroplasma disease prevention and control.

The Eriocheir sinensis calcium/calmodulin-dependent protein kinase II activates apoptosis to resist Spiroplasma eriocheiris infection

Calcium/calmodulin-dependent protein kinase II is a downstream mediator of calcium signalling and participates in the regulation of various cellular physiological functions. In previous studies, the expression of Eriocheir sinensis CaMKII (EsCaMKII) was significantly decreased in the thoracic ganglion after Spiroplasma eriocheiris infection, as shown using TMT-based quantitative proteomic analysis; however, the specific functions of EsCaMKII are still unclear. In this study, the full-length cDNA of EsCaMKII was 3314 bp long, consisting of a 1605 bp open reading frame encoding a protein of 535 amino acids, including a 258 aa serine/threonine protein kinase catalytic domain (EsCaMKII-CD). EsCaMKII is highly transcribed in haemocytes, nerves (thoracic ganglion), gills, and muscles, but lowly transcribed in the hepatopancreas, heart, and intestines. The transcription levels of EsCaMKII were altered in E. sinensis haemocytes after S. eriocheiris infection. After the over-expression of EsCaMKII-CD in RAW264.7 cells, the apoptosis rate of RAW264.7 cells was significantly increased. After the over-expression of EsCaMKII-CD, the morphology of RAW264.7 cells became worse after being infected with S. eriocheiris. Meanwhile, the copy number of S. eriocheiris in RAW264.7 cells was significantly decreased. From 48 h to 96 h after EsCaMKII RNA interference, the transcription levels of EsCaMKII decreased significantly. The transcription of apoptosis genes and cell apoptosis were also inhibited in haemocytes after EsCaMKII RNAi. The knockdown of EsCaMKII by RNAi resulted in significant increases in the copy number of S. eriocheiris and in the mortality of crabs during S. eriocheiris infection. These results indicate that EsCaMKII could promote the apoptosis of E. sinensis and enhance its ability to resist S. eriocheiris infection.

Integrated transcriptome analysis of immune-related mRNAs and microRNAs in Macrobrachium rosenbergii infected with Spiroplasma eriocheiris

Macrobrachium rosenbergii (M. rosenbergii), is a major aquaculture species in China and Southeast Asia. However, infection with Spiroplasma eriocheiris (S. eriocheiris) has caused huge economic losses to the cultivation of M. rosenbergii. Currently, there are few reports on the immune response mechanism of M. rosenbergii that are infected with S. eriocheiris. To clarify the immune response mechanism of M. rosenbergii infected with S. eriocheiris, the key immune genes which respond to the infection with the pathogen and the regulation of related microRNAs (miRNAs) on them were identified. In this study, the mRNA and miRNA transcriptome of hepatopancreas of M. rosenbergii at different infection stages were analyzed using high-throughput sequencing and qRT-PCR. In the mRNA transcriptome, 27,703 and 33,402 genes were expressed in healthy and susceptible M. rosenbergii, respectively. By digital gene-expression profiling analysis, 23,929 and 24,325 genes were expressed, and 223 and 373 genes were significantly up-regulated and down-regulated, respectively. A total of 145 key genes related to Toll, IMD, JAK/STAT and MAPK were excavated from the transcriptome. In the miRNA transcriptome, 549 miRNAs (Conserved: 41, PN-type: 83, PC-type: 425) were sequenced, of which 87 were significantly up-regulated and 23 were significantly down-regulated. Among the related immune pathways, there are 259 miRNAs involved in the regulation of target genes in the Toll and IMD pathways, 231 JAK/STAT pathways and 122 MAPK pathways. qRT-PCR differential detection of immune-related miRNAs and mRNAs showed that 22 miRNAs with significant differences (P < 0.05) such as mro-miR-100, PC-mro-3p-27 and PN-mro-miR-316 had corresponding regulatory relationships with 22 important immune genes such as TLR2, TLR3, TLR4, TLR5, MyD88, Pelle and Relish in different stages after infection. In this study, the immune genes and related regulatory miRNAs of M. rosenbergii in response to S. eriocheiris infection were obtained. The results can provide basic data to further reveal the immune defense mechanism of M. rosenbergii against S. eriocheiris infection.

Transcriptome in combination with experimental validation unveils hub immune-related genes in oriental river prawn Macrobrachium nipponense against Spiroplasma eriocheiris challenge

Spiroplasma eriocheiris (S. eriocheiris) infection has caused heavy economic losses in Macrobrachium nipponense (M. nipponense) aquaculture industry. However, the interplay between immune responses of M. nipponense and S. eriocheiris infection has not yet been fully elucidated. Herein, full-length transcriptome profiling of M. nipponense hemocytes was performed with or without S. eriocheiris challenge. In total, 20,441 high quality unigenes were obtained, among which 733 differentially expressed genes (DEGs) were found, including 309 up-regulated and 424 down-regulated genes. Protein-protein interaction network (PPI) analysis of DEGs identified 4 hub immune-related genes, including MAP kinase-interacting serine/threonine-protein kinase 1 (MNK1), C-type lectin 4 (CTL4), gamma-interferon-inducible lysosomal thiol reductase (GILT), and anti-lipopolysaccharide factor (ALF). Tissue distribution and expression analysis of the key immune-associated genes demonstrated that they were widely expressed in 6 tissues, especially in gills, and were up-regulated by S. eriocheiris challenge. M. nipponense challenged by S. eriocheiris exhibited a significant reduction of bacterial clearance ability when the hub genes were silenced via RNA interference. Overall, our results indicated that the 4 hub immune-related genes play a vital role in anti-S. eriocheiris infection, thus providing insights into the immune defensive response mechanisms of M. nipponense against S. eriocheiris infection.

The cellobiose and α-ketoglutarate as metabolic modulators in Eriocheir sinensis to resist Spiroplasma eriocheiris infection

As one of the most devastating diseases in Eriocheir sinensis, tremor disease (TD) is caused by Spiroplasma eriocheiris and dramatically increases the economic losses of crab aquaculture. With the development of biotechnology, metabolomics plays an important role in the screening of metabolites related to physiological and pathological changes. In this study, the different metabolites induced by S. eriocheiris were investigated in hemocytes of crab E. sinensis using metabolomics based on GC/MS among diverse phenotypes groups (tremor group, no-symptom group, and control group). Compared with the tremor group, 42 differential metabolites (26 up-regulated and 16 down-regulated) were significantly different in the no-symptom group. The KEGG pathway enrichment analysis showed that the differential metabolites were enriched in “ABC transporters”, “Vitamin B6 metabolism”, “Ascorbate and aldarate metabolism” and “Starch and sucrose metabolism”. The cellobiose and α-ketoglutarate (AKG) were chosen to verify the function of these two metabolites. The crabs were fed with dietary cellobiose (1, 5, and 10 mg/g diet weight) or AKG (1, 5, and 10 mg/g diet weight) before S. eriocheiris injection. The results show that all three concentrations of cellobiose and AKG could reduce the mortality of crabs and decrease the copies of S. eriocheiris in the hemocytes. The histopathological sections of the hindgut of E. sinensis show that cellobiose and AKG affect the connective tissue of the outer layer of muscle. Overall, these results provide insight into TD control and the sustainable development of aquaculture.

Full-length transcriptome and long non-coding RNA profiling of whiteleg shrimp Penaeus vannamei hemocytes in response to Spiroplasma eriocheiris infection

Spiroplasma eriocheiris (S. eriocheiris) infection causes a significant economic loss in Penaeus vannamei (P. vannamei) culture industry. However, the response of P. vannamei hemocytes to S. eriocheiris infection has not been extensively studied. In this study, we conducted full-length transcriptome and long non-coding RNA (lncRNA) analyses of P. vannamei hemocytes by a challenge test with S. eriocheiris. Following assembly and annotation, there were 8077 high-quality unigenes. A total of 1168 differentially expressed genes (DEGs) were obtained, including 792 up-regulated and 376 down-regulated genes by differential expression analysis. Gene ontology (GO) enrichment analysis showed that the up-regulated DEGs were mainly clustered into immune system process, defense response, cell cycle and organelle organization. On the other hand, the down-regulated DEGs included that genes that were mainly clustered into metabolic processes related to organic compounds, metabolic process and cellular metabolic process. Protein-protein interaction (PPI) network analysis of DEGs indicated that the pivotal gene interactions were connected to stress response, immune system process and cell cycle. The lncRNA analysis identified multiple lncRNAs, which were highly co-expressed with the immune-related genes, such as lncRNA transcript-12631 and transcript-12631, suggesting that lncRNAs may be involved in the regulation of immune defense in shrimp hemocytes. Additionally, 20 hub unigenes and putative lncRNAs related to immune system were validated by quantitative real-time PCR (qRT-PCR), validating the reliability of RNA-Seq. This study revealed a close connection between the immune and metabolic systems of S. eriocheiris infected P. vannamei.

An ShK-domain serine protease of Eriocheir sinensis regulates the PO activity to resist Spiroplasma eriocheiris infection

A novel serine protease contains two ShK-domain was found from the Chinese mitten crab Eriocheir sinensis (EsShK-SP). The full-length EsShK-SP cDNA is 1927 bp and contains a 1260-bp open reading frame encoding a protein of 420 amino acids, including a signal peptide, two ShK domain, and Tryp-SPC domain. Quantitative real-time PCR showed that EsShK-SP was expressed mainly in the hemocytes, gills, intestine, and nerve, but weakly in heart, muscle, and hepatopancreas. After infected with Spiroplasma eriocheiris, the expression of EsShK-SP was significantly up-regulated from 1 d to 9 d. The Tryp-SPC domain was ligated with pGEX-4T-1 vector and prokaryotic expressed to obtain recombinant protein rSPC. When rSPC and S. eriocheiris stimulated the hemocytes of E. sinensis, the PO activity was significantly up-regulated. The subcellular localization revealed that recombinant EsShK-SP was mainly located in the cytoplasm of Drosophila S2 cells. Both absolute real-time PCR and confocal laser scanning microscope results showed that over-expression of EsShK-SP in S2 cells could decrease the copy number of S. eriocheiris. Meanwhile, the over-expression of EsShK-SP also increased the PO activity and cell viability of S2 cells. After EsShK-SP RNA interference using dsRNA, the expression levels of proPO and activity of PO decreased significantly from 48 h to 96 h. The knockdown of EsShK-SP by RNAi resulted in the copy number of S. eriocheiris in the EsShK-SP silenced group was significantly increased compared to the control groups during S. eriocheiris infection. Meanwhile, the survival rate of crabs decreased in the EsShK-SP-dsRNA group. The above results indicated that EsShK-SP plays an important immune role during E. sinensis against S. eriocheiris through regulation of the proPO system.

Transcriptome profiling of the Eriocheir sinensis thoracic ganglion under the Spiroplasma eriocheiris challenge

Spiroplasma eriocheiris, has been identified as a novel lethal pathogen of Eriocheir sinensis tremor disease (TD, typically paroxysmal tremors of the pereiopod). The thoracic ganglion of E. sinensis infected by S. eriocheiris was the direct reason of the TD. This study was designed to understand of TD processes in crab and the immune response of thoracic ganglion at the transcriptomic levels. The transcriptomic profiles of thoracic ganglion in the experimental group (S. eriocheiris inject, termed SI) and the control group (R2 medium inject, termed RI) were obtained using Illumina HiSeq 2500. In total, there were 60,159,089 and 47,613,445 high-quality clean reads assembled from SI and RI, respectively. In addition, 39,636 unigenes were obtained with an N50 length of 575 bp. Moreover, by RPKM analysis, a total of 8049 differentially expressed genes (SI vs RI) were detected, including 927 up-regulated and 7122 down-regulated genes. The transcription levels of gene in prophenoloxidase-activating system (proPO), Wnt signaling pathway, phagocytosis, Toll-like system, Ca2+ regulation related process, etc. of SI were significantly changed compare with RI. Finally, 12 selected differentially expressed genes (Serpin, TLR, FZ4, SPI2, Tax1, TLSP, AI, CTLR, Rab11A, LAMP-1, Trx1 and PI3K) were verified using qRT-PCR showed similar tendency with the RNA-seq. The results shown that the proPO, Wnt signaling pathway, phagocytosis, Toll-like system, Ca2+ regulation related process, glutamate signal related process, etc. were involved in the process of the S. eriocheiris infection.

Protein N-glycosylation in the early stage of Spiroplasma eriocheiris infect Eriocheir sinensis

As a novel lethal pathogen of Eriocheir sinensis tremor disease, Spiroplasma eriocheiris, has led into catastrophic economic losses in aquaculture. The hemocytes of E. sinensis is the first target cells of S. eriocheiris. Our study is the first time designed to understanding of the N-glycoproteome dynamics of E. sinensis hemocytes under S. eriocheiris infected at the early phage. In the current study, the N-glycoproteome changes of E. sinensis hemocytes after S. eriocheiris infection were obtained using tandem mass tags (TMT) labeling and affinity enrichment followed by high-resolution Liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) analysis. Using a 1.2-fold change in expression as a physiologically significant benchmark, 167 differentially expressed N-glycosylated proteins were reliably quantified, including 79 up-regulated glycosylated proteins and 88 down-regulated glycosylated proteins subsequented to S. eriocheiris infection. Gene ontology (GO) annotation, protein domain annotation, Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation, subcellular localization annotation and protein-protein interaction network were used to analyze those significantly differently expression proteins shown that many biological process and pathway are participate in S. eriocheiris infected host cell, such as phagocytosis, ECM-receptor interaction, lysosome, prophenoloxidase system, and so on. Six selected different expressed glycosylated proteins were studied the transcription at RNA level by qRT-PCR. Our study could serve as a basis to understand the relationship between E. sinensis and the pathogen S. eriocheiris, and also provide reference to study protein N-glycosylation in other crustaceans.

TMT-based quantitative proteomic analysis of Eriocheir sinensis hemocytes and thoracic ganglion during Spiroplasma eriocheiris infection

Spiroplasma eriocheiris, a novel pathogen of Chinese mitten crab Eriocheir sinensis tremor disease, has led into catastrophic economic losses in aquaculture. S. eriocheiris invaded the hemocytes in the early stage, then invaded nerve tissue and caused typically paroxysmal tremors of pereiopod in the late stage of infection. The purpose of this study was to detect the infection mechanism of hemocytes in the early stage and thoracic ganglion in the late stage of S. eriocheiris infection at the protein level. Hemocytes and thoracic ganglion were collected at 24 h and 10 d after injection (the crabs with typical paroxysmal tremors of the pereiopod), respectively. TMT was performed with isobaric markers, followed by liquid chromatography tandem mass spectrometry (LC-MS/MS). In hemocytes, 127 proteins were up-regulated and 85 proteins were down-regulated in 2747 quantified proteins. Many proteins and process including proPO system proteins, hemolymph coagulation system proteins and lectins were differently expressed in hemocytes and involved in the early immune process of E. sinensis against S. eriocheiris infection. Meanwhile, 545 significantly different expression proteins (292 down-regulated and 253 up-regulated protein including a number of immune-associated, nervous system development and signal transmission related proteins) were identified in thoracic ganglion in the late stage of S. eriocheiris infection. The qRT-PCR analysis results shown that the selected significantly changed proteins in hemocytes and thoracic ganglion were consistent with the TMT proteomics. This paper reported for the first time to study the responses of crab hemocyte and thoracic ganglion against the S. eriocheiris infection at different stages. These findings help us understand the infection mechanism of S. eriocheiris at different stage with the different tissue.